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1
Brindley, David N., Abdelkarim Abousalham, Yutaka Kikuchi, Chuen-Neu Wang, and David W. Waggoner. ""Cross talk" between the bioactive glycerolipids and sphingolipids in signal transduction." Biochemistry and Cell Biology 74, no. 4 (July 1, 1996): 469–76. http://dx.doi.org/10.1139/o96-051.
Повний текст джерелаАнотація:
Hydrolysis of phosphatidylcholine via receptor-mediated stimulation of phospholipase D produces phosphatidate that can be converted to lysophosphatidate and diacylglycerol. Diacylglycerol is an activator of protein kinase C, whereas phosphatidate and lysophosphatidate stimulate tyrosine kinases and activate the Ras–Raf–mitogen-activated protein kinase pathway. These three lipids can stimulate cell division. Conversely, activation of sphingomyelinase by agonists (e.g., tumor necrosis factor-α) causes ceramide production that inhibits cell division and produces apoptosis. If ceramides are metabolized to sphingosine and sphingosine 1-phosphate, then these lipids can stimulate phospholipase D and are also mitogenic. By contrast, ceramides inhibit the activation of phospholipase D by decreasing its interaction with the G-proteins, ARF and Rho, which are necessary for its activation. In whole cells, ceramides also stimulate the degradation of phosphatidate, lysophosphatidate, ceramide 1-phosphate, and sphingosine 1-phosphate through a multifunctional phosphohydrolase (the Mg2+-independent phosphatidate phosphohydrolase), whereas sphingosine inhibits phosphatidate phosphohydrolase. Tumor necrosis factor-α causes insulin resistance, which may be partly explained by ceramide production. Cell-permeable ceramides decrease insulin-stimulated glucose uptake in 3T3-L1 adipocytes after 2–24 h, whereas they stimulate basal glucose uptake. These effects do not depend on decreased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 or the interaction of insulin receptor substrate-1 with phosphatidylinositol 3-kinase. They appear to rely on the differential effects of ceramides on the translocation of GLUT1- and GLUT4-containing vesicles. It is concluded that there is a significant interaction and "cross-talk" between the sphingolipid and glycerolipid pathways that modifies signal transduction to control vesicle movement, cell division, and cell death.Key words: ceramides, insulin resistance, phosphatidate, phospholipases, signal transduction.
2
Allende, Maria L., Teiji Sasaki, Hiromichi Kawai, Ana Olivera, Yide Mi, Gerhild van Echten-Deckert, Richard Hajdu, et al. "Mice Deficient in Sphingosine Kinase 1 Are Rendered Lymphopenic by FTY720." Journal of Biological Chemistry 279, no. 50 (September 30, 2004): 52487–92. http://dx.doi.org/10.1074/jbc.m406512200.
Повний текст джерелаАнотація:
Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1.Sphk1null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in mostSphk1-/-tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from theSphk1-/- mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs ofSphk1-/- mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in theSphk1null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with theseSphk1null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.
3
Urtz, Nicole, Ana Olivera, Elisa Bofill-Cardona, Robert Csonga, Andreas Billich, Diana Mechtcheriakova, Frederic Bornancin, Max Woisetschläger, Juan Rivera та Thomas Baumruker. "Early Activation of Sphingosine Kinase in Mast Cells and Recruitment to FcεRI Are Mediated by Its Interaction with Lyn Kinase". Molecular and Cellular Biology 24, № 19 (1 жовтня 2004): 8765–77. http://dx.doi.org/10.1128/mcb.24.19.8765-8777.2004.
Повний текст джерелаАнотація:
ABSTRACT Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca2+, released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-γ and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcεRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcεRI triggering, enhanced sphingosine kinase activity was associated with FcεRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn−/ − mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcεRI proximal event.
4
Dygas, A., M. Sidorko, M. Bobeszko, and J. Barańska. "Exogenous sphingosine 1-phosphate and sphingosylphosphorylcholine do not stimulate phospholipase D in C6 glioma cells." Acta Biochimica Polonica 46, no. 1 (March 31, 1999): 99–106. http://dx.doi.org/10.18388/abp.1999_4187.
Повний текст джерелаАнотація:
In the present study we investigate the effect of exogenous sphingosine, sphingosine 1-phosphate and sphingosylphosphorylcholine on phospholipase D (PLD) activity in glioma C6 cells. The cells were prelabeled with [1-14C]palmitic acid and PLD-mediated synthesis of [14C]phosphatidylethanol was measured. Sphingosine 1-phosphate and sphingosylphosphorylcholine did not stimulate [14C]phosphatidylethanol formation either at low (0.1-10 microM) or high (25-100 microM) concentrations. On the other hand, sphingosine at concentrations of 100-250 microM strongly stimulated PLD activity as compared to the effect of phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), known as a PLD activator. The effect of TPA on PLD is linked to the activation of protein kinase C. The present study also shows that sphingosine additively enhances TPA-mediated PLD activity. This is in contrast to the postulated role of sphingosine as a protein kinase C inhibitor. These results demonstrate that in glioma C6 cells sphingosine not only affects PLD independently of its effect on protein kinase C, but also is unable to block TPA-mediated PLD activity.
5
El-Shewy, Hesham M., Souzan A. Abdel-Samie, Abdelmohsen M. Al Qalam, Mi-Hye Lee, Kazuyuki Kitatani, Viviana Anelli, Ayad A. Jaffa, Lina M. Obeid та Louis M. Luttrell. "Phospholipase C and Protein Kinase C-β 2 Mediate Insulin-Like Growth Factor II-Dependent Sphingosine Kinase 1 Activation". Molecular Endocrinology 25, № 12 (1 грудня 2011): 2144–56. http://dx.doi.org/10.1210/me.2011-0101.
Повний текст джерелаАнотація:
Abstract We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.
6
PYNE, Susan, and Nigel J. PYNE. "Sphingosine 1-phosphate signalling in mammalian cells." Biochemical Journal 349, no. 2 (July 10, 2000): 385–402. http://dx.doi.org/10.1042/bj3490385.
Повний текст джерелаАнотація:
Sphingosine 1-phosphate is formed in cells in response to diverse stimuli, including growth factors, cytokines, G-protein-coupled receptor agonists, antigen, etc. Its production is catalysed by sphingosine kinase, while degradation is either via cleavage to produce palmitaldehyde and phosphoethanolamine or by dephosphorylation. In this review we discuss the most recent advances in our understanding of the role of the enzymes involved in metabolism of this lysolipid. Sphingosine 1-phoshate can also bind to members of the endothelial differentiation gene (EDG) G-protein-coupled receptor family [namely EDG1, EDG3, EDG5 (also known as H218 or AGR16), EDG6 and EDG8] to elicit biological responses. These receptors are coupled differentially via Gi, Gq, G12/13 and Rho to multiple effector systems, including adenylate cyclase, phospholipases C and D, extracellular-signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase and non-receptor tyrosine kinases. These signalling pathways are linked to transcription factor activation, cytoskeletal proteins, adhesion molecule expression, caspase activities, etc. Therefore sphingosine 1-phosphate can affect diverse biological responses, including mitogenesis, differentiation, migration and apoptosis, via receptor-dependent mechanisms. Additionally, sphingosine 1-phosphate has been proposed to play an intracellular role, for example in Ca2+ mobilization, activation of non-receptor tyrosine kinases, inhibition of caspases, etc. We review the evidence for both intracellular and extracellular actions, and extensively discuss future approaches that will ultimately resolve the question of dual action. Certainly, sphingosine 1-phosphate will prove to be unique if it elicits both extra- and intra-cellular actions. Finally, we review the evidence that implicates sphingosine 1-phosphate in pathophysiological disease states, such as cancer, angiogenesis and inflammation. Thus there is a need for the development of new therapeutic compounds, such as receptor antagonists. However, identification of the most suitable targets for drug intervention requires a full understanding of the signalling and action profile of this lysosphingolipid. This article describes where the research field is in relation to achieving this aim.
7
Desai, N. N., R. O. Carlson, M. E. Mattie, A. Olivera, N. E. Buckley, T. Seki, G. Brooker, and S. Spiegel. "Signaling pathways for sphingosylphosphorylcholine-mediated mitogenesis in Swiss 3T3 fibroblasts." Journal of Cell Biology 121, no. 6 (June 15, 1993): 1385–95. http://dx.doi.org/10.1083/jcb.121.6.1385.
Повний текст джерелаАнотація:
Sphingosylphosphorylcholine (SPC), or lysophingomyelin, a wide-spectrum growth promoting agent for a variety of cell types (Desai, N. N., and S. Spiegel. 1991. Biochem. Biophys. Res. Comm. 181: 361-366), stimulates cellular proliferation of quiescent Swiss 3T3 fibroblasts to a greater extent than other known growth factors or than the structurally related molecules, sphingosine and sphingosine-1-phosphate. SPC potentiated the mitogenic effect of an activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate, and did not compete with phorbol esters for binding to protein kinase C in intact Swiss 3T3 fibroblasts. However, downregulation of protein kinase C, by prolonged treatment with phorbol ester, reduced, but did not eliminate, the ability of SPC to stimulate DNA synthesis, indicating that SPC may act via both protein kinase C-dependent and -independent signaling pathways. SPC induced a rapid rise in intracellular free calcium ([Ca2+]i) in viable 3T3 fibroblasts determined with a digital imaging system. Although the increases in [Ca2+]i were observed even in the absence of calcium in the external medium, no increase in the levels of inositol phosphates could be detected in response to mitogenic concentrations of SPC. Furthermore, in contrast to sphingosine or sphingosine-1-phosphate, the mitogenic effect of SPC was not accompanied by increases in phosphatidic acid levels or changes in cAMP levels. SPC, but not sphingosine or sphingosine-1-phosphate, stimulates the release of arachidonic acid. Therefore, the ability of SPC to act an extremely potent mitogen may be due to activation of signaling pathway(s) distinct from those used by sphingosine or sphingosine-1-phosphate.
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King, Charles C., Frank T. Zenke, Philip E. Dawson, Erica M. Dutil, Alexandra C. Newton, Brian A. Hemmings, and Gary M. Bokoch. "Sphingosine Is a Novel Activator of 3-Phosphoinositide-dependent Kinase 1." Journal of Biological Chemistry 275, no. 24 (March 29, 2000): 18108–13. http://dx.doi.org/10.1074/jbc.m909663199.
Повний текст джерела
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Spertini, F., A. V. Wang, T. Chatila, and R. S. Geha. "Engagement of the common leukocyte antigen CD45 induces homotypic adhesion of activated human T cells." Journal of Immunology 153, no. 4 (August 15, 1994): 1593–602. http://dx.doi.org/10.4049/jimmunol.153.4.1593.
Повний текст джерелаАнотація:
Abstract The transmembrane tyrosine phosphatase CD45 plays an important role in TCR/CD3-mediated signaling. We demonstrate in this study that ligand binding to the CD45 molecule induces homotypic cell adhesion of activated, but not resting, T lymphocytes. mAbs to CD45 (4B2 and 10G10) and to CD45RO (UCHL1), but not to CD45RA (IOL2), caused sustained adhesion of alloreactive T cell lines. In contrast, none of the anti-CD45 mAbs induced aggregation of resting peripheral T cells. CD45-mediated adhesion of activated T cells involved both CD11a/18-dependent as well as CD11a/18-independent mechanisms. mAb 4B2 induced a strictly CD11a/18-dependent adhesion that was completely inhibited by both the protein kinase C (PKC) inhibitor sphingosine and the protein tyrosine kinase (PTK) inhibitors genestein and herbimycin A. In contrast, mAb 10G10, which recognized an epitope on CD45 distinct from the one recognized by mAb 4B2, induced CD11a/18-independent adhesion that was inhibited by sphingosine, but not by genestein or herbimycin A. Biochemical studies revealed direct evidence for activation of protein kinase C and protein tyrosine kinase after engagement of CD45 on activated T cells by mAb 4B2. These results indicate that in addition to its role in TCR/CD3-mediated activation, engagement of CD45 transduces signals that result in enhanced adhesiveness of activated T cells.
10
Mourad, W., R. S. Geha, and T. Chatila. "Engagement of major histocompatibility complex class II molecules induces sustained, lymphocyte function-associated molecule 1-dependent cell adhesion." Journal of Experimental Medicine 172, no. 5 (November 1, 1990): 1513–16. http://dx.doi.org/10.1084/jem.172.5.1513.
Повний текст джерелаАнотація:
Antigenic stimulation is associated with enhanced adhesion between T cells and antigen-presenting cells (APC). Binding of ligands to the T cell antigen receptor activates the adhesion function of lymphocyte function-associated molecule 1 (LFA-1; CD11a/CD18). We demonstrate here that ligand binding to major histocompatibility complex class II (Ia) molecules also activates LFA-1 function, providing a reciprocal mechanism for the induction of adhesion between T cells and Ia+ APC. Adhesion was affected by a qualitative change in LFA-1 molecules and was reversed by the protein kinase C inhibitor sphingosine. These results define a novel role for Ia molecules as signal transducing receptors that regulate LFA-1-dependent adhesion via a putative, Ia-coupled protein kinase(s).
11
Aoki, Masayo, Hiroaki Aoki, Partha Mukhopadhyay, Takuya Tsuge, Hirofumi Yamamoto, Noriko M. Matsumoto, Eri Toyohara, Yuri Okubo, Rei Ogawa, and Kazuaki Takabe. "Sphingosine-1-Phosphate Facilitates Skin Wound Healing by Increasing Angiogenesis and Inflammatory Cell Recruitment with Less Scar Formation." International Journal of Molecular Sciences 20, no. 14 (July 10, 2019): 3381. http://dx.doi.org/10.3390/ijms20143381.
Повний текст джерелаАнотація:
Wound healing starts with the recruitment of inflammatory cells that secrete wound-related factors. This step is followed by fibroblast activation and tissue construction. Sphingosine-1-phosphate (S1P) is a lipid mediator that promotes angiogenesis, cell proliferation, and attracts immune cells. We investigated the roles of S1P in skin wound healing by altering the expression of its biogenic enzyme, sphingosine kinase-1 (SphK1). The murine excisional wound splinting model was used. Sphingosine kinase-1 (SphK1) was highly expressed in murine wounds and that SphK1−/− mice exhibit delayed wound closure along with less angiogenesis and inflammatory cell recruitment. Nanoparticle-mediated topical SphK1 overexpression accelerated wound closure, which associated with increased angiogenesis, inflammatory cell recruitment, and various wound-related factors. The SphK1 overexpression also led to less scarring, and the interaction between transforming growth factor (TGF)-β1 and S1P receptor-2 (S1PR2) signaling is likely to play a key role. In summary, SphK1 play important roles to strengthen immunity, and contributes early wound healing with suppressed scarring. S1P can be a novel therapeutic molecule with anti-scarring effect in surgical, trauma, and chronic wound management.
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Limaye, Vidya, Xiaochun Li, Chris Hahn, Pu Xia, Michael C. Berndt, Mathew A. Vadas, and Jennifer R. Gamble. "Sphingosine kinase-1 enhances endothelial cell survival through a PECAM-1–dependent activation of PI-3K/Akt and regulation of Bcl-2 family members." Blood 105, no. 8 (April 15, 2005): 3169–77. http://dx.doi.org/10.1182/blood-2004-02-0452.
Повний текст джерелаАнотація:
AbstractSphingosine-1-phosphate (S1P), the bioactive product of sphingosine kinase (SK) activation, is a survival factor for endothelial cells. The mechanism of SK-mediated survival was investigated in endothelial cells with moderately raised intracellular SK activity. Overexpression of SK mediated survival primarily through the activation of the phosphatidyl inositol 3-kinase (PI-3K)/protein kinase B (Akt/PKB) pathway and an associated up-regulation of the antiapoptotic protein B cell lymphoma gene 2 (Bcl-2) and down-regulation of the proapoptotic protein bisindolylmaleimide (Bcl-2 interacting mediator of cell death; Bim). In addition there was an up-regulation and dephosphorylation of the junctional molecule platelet endothelial cell adhesion molecule-1 (PECAM-1), which was obligatory for activation of the PI-3K/Akt pathway, for SK-induced cell survival, and for the changes in the apoptosis-related proteins. Thus, raised intracellular SK activity induced a molecule involved in cell–cell interactions to augment cell survival through a PI-3K/Akt–dependent pathway. This is distinct from the activation of both PI-3K/Akt and mitogen-activated protein kinase (MAPK) pathways seen with exogenously added S1P. Cells overexpressing SK showed enhanced survival under conditions of serum deprivation and absence of attachment to extracellular matrix, suggesting a role for SK in the regulation of vascular phenomena that occur under conditions of stress, such as angiogenesis and survival in unattached states, as would be required for a circulating endothelial cell.
13
Shamshiddinova, Maftuna, Shokhid Gulyamov, Hee-Jung Kim, Seo-Hyeon Jung, Dong-Jae Baek, and Yong-Moon Lee. "A Dansyl-Modified Sphingosine Kinase Inhibitor DPF-543 Enhanced De Novo Ceramide Generation." International Journal of Molecular Sciences 22, no. 17 (August 25, 2021): 9190. http://dx.doi.org/10.3390/ijms22179190.
Повний текст джерелаАнотація:
Sphingosine-1-phosphate (S1P) synthesized by sphingosine kinase (SPHK) is a signaling molecule, involved in cell proliferation, growth, differentiation, and survival. Indeed, a sharp increase of S1P is linked to a pathological outcome with inflammation, cancer metastasis, or angiogenesis, etc. In this regard, SPHK/S1P axis regulation has been a specific issue in the anticancer strategy to turn accumulated sphingosine (SPN) into cytotoxic ceramides (Cers). For these purposes, there have been numerous chemicals synthesized for SPHK inhibition. In this study, we investigated the comparative efficiency of dansylated PF-543 (DPF-543) on the Cers synthesis along with PF-543. DPF-543 deserved attention in strong cytotoxicity, due to the cytotoxic Cers accumulation by ceramide synthase (CerSs). DPF-543 exhibited dual actions on Cers synthesis by enhancing serine palmitoyltransferase (SPT) activity, and by inhibiting SPHKs, which eventually induced an unusual environment with a high amount of 3-ketosphinganine and sphinganine (SPA). SPA in turn was consumed to synthesize Cers via de novo pathway. Interestingly, PF-543 increased only the SPN level, but not for SPA. In addition, DPF-543 mildly activates acid sphingomyelinase (aSMase), which contributes a partial increase in Cers. Collectively, a dansyl-modified DPF-543 relatively enhanced Cers accumulation via de novo pathway which was not observed in PF-543. Our results demonstrated that the structural modification on SPHK inhibitors is still an attractive anticancer strategy by regulating sphingolipid metabolism.
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He, H., AC Herington, and P. Roupas. "Effect of protein kinase C modulation on gonadotrophin-induced granulosa cell steroidogenesis." Reproduction, Fertility and Development 7, no. 1 (1995): 83. http://dx.doi.org/10.1071/rd9950083.
Повний текст джерелаАнотація:
The effect of protein kinase C (PKC) modulation on gonadotrophin-induced ovarian granulosa cell differentiation was investigated by using an activator of PKC, phorbol 12-myristate 13-acetate (PMA) and inhibitors of PKC, sphingosine (SPH) and staurosporine (ST). The effects of PMA (at doses which activate PKC (10 ng mL-1), and down-regulate PKC (1000 ng mL-1)), sphingosine (25 microM) and staurosporine (10(-10)-10(-7) M) on gonadotrophin-induced granulosa cell differentiation were studied by the determination of steroidogenesis and cAMP accumulation in immature rat ovarian granulosa cells treated with or without pregnant mare serum gonadotrophin (100 mU mL-1). PMA (10 ng mL-1) inhibited gonadotrophin-induced granulosa cell steroidogenesis and cAMP accumulation. PMA (1000 ng mL-1)-induced down-regulation of PKC did not affect gonadotrophin-induced steroidogenesis. The inhibitory effect of PMA (10 ng mL-1) on gonadotrophin-induced granulosa cell steroidogenesis was not present in PKC-down-regulated cells. These data indicate that PKC activation by PMA inhibits gonadotrophin-induced steroidogenesis. SPH also inhibited gonadotrophin-induced steroidogenesis and cAMP accumulation. This effect of SPH was not affected by PMA-induced PKC down-regulation, indicating that this action of SPH does not require PKC or is mediated via a phorbol ester-insensitive PKC isoform. ST induced steroidogenesis in the absence of gonadotrophin, but was not synergistic with gonadotrophin. PMA-induced down-regulation of PKC abolished the effect of ST, suggesting that the action of ST requires PKC. The data suggest that ST and PMA, which antagonize each other in gonadotrophin-induced steroidogenesis, act via a PKC-mediated mechanism whereas the cAMP-associated actions of gonadotrophins and SPH are not dependent on PKC.
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Newton, Jason, Elisa N. D. Palladino, Cynthia Weigel, Michael Maceyka, Markus H. Gräler, Can E. Senkal, Ricardo D. Enriz, et al. "Targeting defective sphingosine kinase 1 in Niemann–Pick type C disease with an activator mitigates cholesterol accumulation." Journal of Biological Chemistry 295, no. 27 (May 8, 2020): 9121–33. http://dx.doi.org/10.1074/jbc.ra120.012659.
Повний текст джерелаАнотація:
Niemann–Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism.
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Melendez, Alirio J., Manikandan Jayapal, and Peter Natesan Pushparaj. "SIRNA KNOCKDOWN OF SPHK1 PROTECTS MICE FROM IGE-MEDIATED ANAPHYLAXIS (37.6)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S19. http://dx.doi.org/10.4049/jimmunol.178.supp.37.6.
Повний текст джерелаАнотація:
Abstract Sphingosine kinases are emerging a key enzymes in the intracellular signaling cascades, that mediate inflammatory responses, triggered by various inflammatory stimuli including: anaphylatoxins (C5a); bacterial-derived peptides (fMLP); and IgG or IgE receptor stimulation (FcγRI and FcεRI), in various immune cells, including in human mast cells. Moreover, we have recently reported a study on genome-wide gene expression profile of human mast cells sensitized with IgE alone, or stimulated by FcεRI aggregation, and found that sphingosine kinase 1 (SphK1) is one of the early genes to be activated. This, coupled to our previous studies indicating that SphK1 is pivotal in the signaling pathways initiated by FcεRI in human mast cells, prompted us to investigate the functional role of SphK1 in IgE-triggered inflammation/anaphylaxis in vivo. The systemic anaphylaxis reaction in mice has been considered to be a typical immediate hypersensitivity response determined by the activation of immune cells via antigen-induced aggregation of an IgE-sensitized FcεRI. To this end, we silenced the SPHK1 gene in vivo (using a specific siRNA), and investigated whether SphK1 knockdown influenced the outcome of the acute inflammatory responses, triggered during IgE-mediated systemic anaphylaxis in mice. We show here that, the siRNA for SphK1 protected mice from the IgE-mediated allergic reactions, including inhibition on: temperature changes; vascular permeability; immune-cell infiltration into organs; cytokine production; and vascular cell adhesion molecule expression. Thus, our findings strongly support a role for SpkK1 IgE-mediated anaphylaxis in vivo.
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Zhang, Jianqing, Norman Honbo, Edward J. Goetzl, Kanu Chatterjee, Joel S. Karliner, and Mary O. Gray. "Signals from type 1 sphingosine 1-phosphate receptors enhance adult mouse cardiac myocyte survival during hypoxia." American Journal of Physiology-Heart and Circulatory Physiology 293, no. 5 (November 2007): H3150—H3158. http://dx.doi.org/10.1152/ajpheart.00587.2006.
Повний текст джерелаАнотація:
Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Immune stimuli increase S1P synthesis and secretion by mast cells and platelets, implicating this molecule in tissue responses to injury and inflammation. Binding of S1P to Gi protein-coupled receptors activates phosphatidylinositol 3-kinase and Akt in a variety of tissues. To elucidate the mechanisms by which S1P enhances adult cardiac myocyte survival during hypoxia, we used a mouse cell culture system in which S1P1 receptors were observed to transduce signals from exogenous S1P, an S1P1 receptor antibody with agonist properties, and the pharmacological agents FTY720 and SEW2871. S1P1 receptor mRNA and protein were abundantly expressed by adult mouse cardiac myocytes. S1P-S1P1 receptor axis enhancement of myocyte survival during hypoxia was abolished by phosphatidylinositol 3-kinase inhibition. S1P1 receptor function was closely associated with activation of Akt, inactivation of GSK-3β, and reduction of cytochrome c release from heart mitochondria. These observations highlight the importance of S1P1 receptors on ventricular myocytes as mediators of inducible resistance against cellular injury during severe hypoxic stress.
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Park, Hwa-young, Jong-Kyung Youm, Mi Jung Kwon, Byeong Deog Park, Seung Hun Lee, and Eung Ho Choi. "K6PC-5, a novel sphingosine kinase activator, improves long-term ultraviolet light-exposed aged murine skin." Experimental Dermatology 17, no. 10 (October 2008): 829–36. http://dx.doi.org/10.1111/j.1600-0625.2008.00708.x.
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Giusto, Kiersten, and Charles R. Ashby. "Investigating the Et-1/SphK/S1P Pathway as a Novel Approach for the Prevention of Inflammation-Induced Preterm Birth." Current Pharmaceutical Design 24, no. 9 (May 18, 2018): 983–88. http://dx.doi.org/10.2174/1381612824666180130122739.
Повний текст джерелаАнотація:
Background: Preterm birth (PTB), defined as birth before 37 completed weeks of gestation, occurs in up to 18 percent of births worldwide and accounts for the majority of perinatal morbidity and mortality. While the single most common cause of PTB has been identified as inflammation, safe and effective pharmacotherapy to prevent PTB has yet to be developed. Methods: Our group has used an in vivo model of inflammation-driven PTB, biochemical methods, pharmacological approaches, a novel endothelin receptor antagonist that we synthesized and RNA knockdown to help establish the role of endothelin-1 (ET-1) in inflammation-associated PTB. Further, we have used our in vivo model to test whether sphingosine kinase, which acts downstream of ET-1, plays a role in PTB. Results: We have shown that levels of endothelin converting enzyme-1 (ECE-1) and ET-1 are increased when PTB is induced in timed pregnant mice with lipopolysaccharide (LPS) and that blocking ET-1 action, pharmacologically or using ECE-1 RNA silencing, rescues LPS-induced mice from PTB. ET-1 activates the sphingosine kinase/sphingosine-1-phosphate (SphK/S1P) pathway. S1P, in turn, is an important signaling molecule in the proinflammatory response. Interestingly, we have shown that SphK inhibition also prevents LPS-induced PTB in timed pregnant mice. Further, we showed that SphK inhibition suppresses the ECE-1/ET-1 axis, implicating positive feedback regulation of the SphK/S1P/ECE-1/ET-1 axis. Conclusion: The ET-1/SphK/SIP pathway is a potential pharmacotherapeutic target for the prevention of PTB.
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Kleger, Alexander, Stefan Liebau, Qiong Lin, Götz von Wichert, and Thomas Seufferlein. "The Impact of Bioactive Lipids on Cardiovascular Development." Stem Cells International 2011 (2011): 1–13. http://dx.doi.org/10.4061/2011/916180.
Повний текст джерелаАнотація:
Lysophospholipids comprise a group of bioactive molecules with multiple biological functions. The cardinal members of this signalling molecule group are sphingosylphosphorylcholine (SPC), lysophosphatidic acid (LPA), and sphingosine 1-phosphate (S1P) which are, at least in part, homologous to each other. Bioactive lipids usually act via G-protein coupled receptors (GPCRs), but can also function as direct intracellular messengers. Recently, it became evident that bioactive lipids play a role during cellular differentiation development. SPC induces mesodermal differentiation of mouse ES cells and differentiation of promyelocytic leukemia cells, by a mechanism being critically dependent on MEK-ERK signalling. LPA stimulates the clonal expansion of neurospheres from neural stem/progenitor cells and induces c-fos via activation of mitogen- and stress-activated protein kinase 1 (MSK1) in ES cells. S1P acts on hematopoietic progenitor cells as a chemotactic factor and has also been found to be critical for cardiac and skeletal muscle regeneration. Furthermore, S1P promotes cardiogenesis and similarly activates Erk signalling in mouse ES cells. Interestingly, S1P may also act to maintain human stem cell pluripotency. Both LPA and S1P positively regulate the proliferative capacity of murine ES cells. In this paper we will focus on the differential and developmental impact of lysophospholipids on cardiovascular development.
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Caruso-Neves, C., J. R. Meyer-Fernandes, and A. G. Lopes. "Osmotic Modulation of the Ouabain-Sensitive (Na++K+)ATPase from Malpighian Tubules of Rhodnius prolixus." Zeitschrift für Naturforschung C 53, no. 9-10 (October 1, 1998): 911–17. http://dx.doi.org/10.1515/znc-1998-9-1021.
Повний текст джерелаАнотація:
The presence and regulation by hyperosmotic medium of the ouabain-sensitive (Na++K+)ATPase of the Malpighian tubule cells of Rhodnius prolixus was investigated. The ouabain-sensitive (Na++K+)ATPase activity was 5.4 ± 0.5 nmol Pi x mg-1 x min-1. Vanadate 100 μM completely abolished this ATPase activity. In hyperosmotic medium, obtained by addition of 180 mᴍ mannitol, the (Na++K+)ATPase activity was inhibited by 60%. When the cell lysates were preincubated in hyperosmotic medium for 30 minutes and the ATPase activity was assayed in isosmotic medium, the (Na++K+)ATPase activity was not modified. Addition of 50 ng/ml sphingosine, a protein kinase C inhibitor, abolished the inhibition of (Na++K+)ATPase activity in hyperosmotic medium. Furtherm ore, phorbol ester (TPA), an activator of protein kinase C, mimicked the effect of hyperosmotic shock on (Na++K+)ATPase activity. The increase in Ca2+ concentration decreased the (Na++K+)ATPase activity by 60% in isosmotic medium, with maximal effect obtained in 10-6 m Ca2+. No effect was observed in hyperosmotic medium. The inhibitory effect of Ca2+ on the (Na++K+)ATPase was not reversed by sphingosine. These results indicate that the ouabain-sensitive (Na++K+) ATPase activity of the Malpighian tubule is regulated by both increasing Ca2+ concentration and by the osmolality of the medium by different and integrative ways.
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Katkade, Vaibhav, Abigail A. Soyombo, Irma Isordia-Salas, Harlan N. Bradford, John P. Gaughan, Robert W. Colman, and Tracee S. Panetti. "Domain 5 of cleaved high molecular weight kininogen inhibits endothelial cell migration through Akt." Thrombosis and Haemostasis 94, no. 09 (2005): 606–14. http://dx.doi.org/10.1160/th04-12-0834.
Повний текст джерелаАнотація:
SummaryDomain 5 (D5) of cleaved high molecular weight kininogen (HKa) inhibits angiogenesis in vivo and endothelial cell migration in vitro, but the cell signaling pathways involved in HKa and D5 inhibition of endothelial cell migration are incompletely delineated. This study examines the mechanism of HKa and D5 inhibition of two potent stimulators of endothelial cell migration, sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF), that act through the PI3-kinase-Akt signaling pathway. HKa and D5 inhibit bovine pulmonary artery endothelial cell (BPAE) or human umbilical vein endothelial cell chemotaxis in the modified-Boyden chamber in response to VEGF or S1P. The inhibition of migration by HKa is reversed by antibodies to urokinase-type plasminogen activator receptor. Both HKa and D5 decrease the speed of BPAE cell migration and alter the morphology in live, time-lapse microscopy after stimulation with S1P or VEGF. HKa and D5 reduce the localization of paxillin to the focal adhesions after S1P and VEGF stimulation. To better understand the intracellular signaling pathways, we examined the effect of HKa on the phosphorylation of Akt and its downstream effector, GSK-3α.HKa and D5 inhibit phosphorylation of Akt and GSK-3α after stimulation withVEGF and S1P. Inhibitors of Akt and PI3-kinase, the upstream activator of Akt, block endothelial cell migration and disrupt paxillin localization to the focal adhesions after stimulation with VEGF and S1P. Therefore we suggest that HKa through its D5 domain alters PI3-kinase- Akt signaling to inhibit endothelial cell migration through alterations in the focal adhesions.
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Youm, Jong-Kyung, Hae Jo, Jeong Hee Hong, Dong Min Shin, Mi Jung Kwon, Se Kyoo Jeong, Byeong Deog Park, Eung Ho Choi, and Seung Hun Lee. "K6PC-5, a sphingosine kinase activator, induces anti-aging effects in intrinsically aged skin through intracellular Ca2+ signaling." Journal of Dermatological Science 51, no. 2 (August 2008): 89–102. http://dx.doi.org/10.1016/j.jdermsci.2008.03.002.
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Sun, Kaiqi, Yujin Zhang, Mikhail Bogdanov, William Dowhan, Modupe Idowu, Harinder S. Juneja, Michael R. Blackburn, Rodney E. Kellems, and Yang Xia. "Elevated Adenosine Signaling Via Adenosine A2B Receptor Induces Normal and Sickle Erythrocyte Sphingosine Kinase 1 Activity." Blood 124, no. 21 (December 6, 2014): 4067. http://dx.doi.org/10.1182/blood.v124.21.4067.4067.
Повний текст джерелаАнотація:
Abstract Sickle Cell Disease (SCD) is one of the most devastating hemolytic genetic disorders affecting millions worldwide. Erythrocytes possess high sphingosine kinase 1 (Sphk1) activity and are considered to be the major cell type for supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is up-regulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte Sphk1 activity is regulated in SCD remains unknown. In an effort to identify specific factors and signaling pathways involved in regulation of erythrocyte SphK1 activities in SCD, we first chose to screen the effects of molecules known to induce SphK1 activities in other cell types and/or reported to be elevated in the circulation of SCD including tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF), S1P and adenosine, on the activities of SphK1 in cultured primary mouse normal erythrocytes. Among all of those molecules tested, we found that adenosine is a previously unidentified hypoxia inducible molecule directly inducing SphK1 activity in vitro in a time and dosage-dependent manner. Next, using four adenosine receptor deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in cultured primary mouse normal and sickle erythrocytes. Subsequently, we provided in vivo genetic evidence that adenosine deaminase (ADA)-deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Mechanistically, we revealed that PKA functions downstream of ADORA2B mediating ERK activation and subsequently underlying adenosine-induced SphK1 activities in cultured mouse erythrocytes. Finally, we conducted human translational studies and reported that adenosine signaling via ADORA2B directly increases SphK1 activity in cultured primary human normal and sickle erythrocytes in a PKA/ERK-dependent manner. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms to control SphK1 activity in normal and sickle setting. Disclosures No relevant conflicts of interest to declare.
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Bianco, Roberto, Roberta Rosa, Lucia Nappi, Luigi Formisano, Vincenzo Damiano, Claudia d'Amato, Ana Paula de Maio, et al. "Role of sphingosine kinase 1 (SphK1) on cetuximab resistance in colorectal cancer models." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): e13509-e13509. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e13509.
Повний текст джерелаАнотація:
e13509 Background: Although EGFR inhibitors, such as the mAb cetuximab, represent an effective strategy in colorectal cancer (CRC), the clinical use of these agents is limited by intrinsic or acquired resistance. Alterations in the ‘sphingolipid rheostat’, or the balance between the proapoptotic molecule ceramide and the mitogenic factor sphingosine-1-phosphate (S1P), due to overactivation of sphingosine kinase 1 (SphK1), have been involved in the regulation of resistance to anticancer agents. Since some studies described cross-talks between SphK1 and EGFR-dependent signalling pathways, we investigated the contribution of SphK1 to cetuximab resistance in CRC models. Methods: We used CRC cell lines with both intrinsic or acquired resistance to cetuximab. In these models, we analyzed SphK1 expression/activation by using different tools, including the available drug fingolimod (FTY720), both in vitro and in vivo. We confirmed our data through a tissue microarray (TMA)-based analysis on CRC tissues. Results: SphK1 is overexpressed in CRC cells resistant to cetuximab. Higher doses of N,N-dimethylsphingosine (DMS), a potent competitive inhibitor of SphK1, are needed to achieve complete enzyme saturation and survival inhibition in resistant cells. Moreover, ceramide induces apoptosis less efficiently in resistant than in sensitive cells, consistently with the idea that increased SphK1 levels mediate S1P synthesis by ceramide in resistant cells. SphK1 contribution to resistance is supported by the demonstration that SphK1 inhibition by DMS or silencing via siRNA in resistant cells restores sensitivity to cetuximab, whereas exogenous SphK1 overexpression in wild-type cells confers resistance. Re-sensitization to cetuximab is observed after treatment with fingolimod, a S1P receptor inhibitor, both in vitro and in nude mice xenografted with CRC cells. Finally, a TMA-based analysis on CRC tissues revealed that SphK1 expression is related to K-Ras mutational status, a well-known determinant of cetuximab resistance. Conclusions: Our data could clarify the role of SphK1 in the onset of resistance to cetuximab, thus suggesting SphK1 inhibition as a part of novel targeting strategies for resistant cancer patients.
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Tokuyama, Michio, and Tomotaka Mabuchi. "New Treatment Addressing the Pathogenesis of Psoriasis." International Journal of Molecular Sciences 21, no. 20 (October 11, 2020): 7488. http://dx.doi.org/10.3390/ijms21207488.
Повний текст джерелаАнотація:
Psoriasis is an immune cell-mediated inflammatory skin disease. The interleukin (IL)23/IL17 axis plays an important role in the development of psoriasis. The effectiveness of biologic treatments such as tumor necrosis factor (TNF)α inhibitors (infliximab, adalimumab, certolizumab pegol), IL23 inhibitors (ustekinumab, guselkumab, tildrakizumab, risankizumab), and IL17 inhibitors (secukinumab, ixekizumab, brodalumab) have verified these findings. Immune-related cells such as dendritic cells (DCs) and macrophages, in addition to Toll-like receptors and cytokines such as interferon (IFN)α, TNFα, IFNɤ, IL12, IL22, IL23, and IL17, are related to the pathogenesis of psoriasis. Here, we first review new insights regarding the pathogenesis of psoriasis, as it relates to DCs, Langerhans cells, macrophages, the signal transducer and activator of transcription 3 pathway, and aryl hydrocarbon receptor in cutaneous vascular endothelial cells. Based on these findings, we summarize currently available oral treatments and biologics. Furthermore, we describe a new treatment option including Janus kinase inhibitor, tyrosine kinase 2 inhibitor, modulator of sphingosine 1-phosphate receptor 1, and Rho-associated kinase 2 inhibitor.
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Downey, G. P., C. K. Chan, P. Lea, A. Takai, and S. Grinstein. "Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C." Journal of Cell Biology 116, no. 3 (February 1, 1992): 695–706. http://dx.doi.org/10.1083/jcb.116.3.695.
Повний текст джерелаАнотація:
The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.
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Li, Donghui, Aarti N. Urs, Jeremy Allegood, Adam Leon, Alfred H. Merrill та Marion B. Sewer. "Cyclic AMP-Stimulated Interaction between Steroidogenic Factor 1 and Diacylglycerol Kinase θ Facilitates Induction of CYP17". Molecular and Cellular Biology 27, № 19 (30 липня 2007): 6669–85. http://dx.doi.org/10.1128/mcb.00355-07.
Повний текст джерелаАнотація:
ABSTRACT In the human adrenal cortex, adrenocorticotropin (ACTH) activates CYP17 transcription by promoting the binding of the nuclear receptor steroidogenic factor 1 (SF1) (Ad4BP, NR5A1) to the promoter. We recently found that sphingosine is an antagonist for SF1 and inhibits cyclic AMP (cAMP)-dependent CYP17 gene transcription. The aim of the current study was to identify phospholipids that bind to SF1 and to characterize the mechanism by which ACTH/cAMP regulates the biosynthesis of this molecule(s). Using tandem mass spectrometry, we show that in H295R human adrenocortical cells, SF1 is bound to phosphatidic acid (PA). Activation of the ACTH/cAMP signal transduction cascade rapidly increases nuclear diacylglycerol kinase (DGK) activity and PA production. PA stimulates SF1-dependent transcription of CYP17 reporter plasmids, promotes coactivator recruitment, and induces the mRNA expression of CYP17 and several other steroidogenic genes. Inhibition of DGK activity attenuates the binding of SF1 to the CYP17 promoter, and silencing of DGK-θ expression inhibits cAMP-dependent CYP17 transcription. LXXLL motifs in DGK-θ mediate a direct interaction of SF1 with the kinase and may facilitate binding of PA to the receptor. We conclude that ACTH/cAMP stimulates PA production in the nucleus of H295R cells and that this increase in PA concentrations facilitates CYP17 induction.
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Qin, Zhiqiang, Lu Dai, Karlie Plaisance-Bonstaff, Can Senkal, Wenxue Wang, Thomas M. Reske, Luis Del Valle, et al. "Targeting Sphingosine Kinase Induces Apoptosis and Regression Of Virus-Associated Lymphoma In Vivo." Blood 122, no. 21 (November 15, 2013): 4414. http://dx.doi.org/10.1182/blood.v122.21.4414.4414.
Повний текст джерелаАнотація:
Background and Specific Aim Sphingosine kinase (SphK) is overexpressed by a variety of cancers, and its phosphorylation of sphingosine results in accumulation of sphingosine-1-phosphate (S1P) and activation of anti-apoptotic signal transduction. Existing data indicate a role for S1P in viral pathogenesis, but roles for SphK and S1P in virus-associated cancer progression have not been defined. The Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of primary effusion lymphoma (PEL)—a rapidly progressive tumor arising in body cavities which incurs a median survival time of around 6 months with standard therapeutic approaches. ABC294640 is an orally bioavailable small molecule inhibitor of SphK under evaluation in early-phase clinical trials, although no pre-clinical or clinical data are available for this agent for hematologic or virus-associated malignancies. Therefore, we sought to determine whether ABC294640 displays inhibitory effects for HIV/KSHV+ patient-derived PEL cells in vitro and in vivo, as well as potential mechanisms through which SphK regulates KSHV pathogenesis. Methodology Complementary in vitro assays were undertaken using RNAi and ABC294640 for targeting SphK1 and SphK2 in HIV/KSHV+ patient-derived PEL cell lines (uninfected B cell tumor lines were used as controls). qRT-PCR and immunoblots were used to quantify KSHV gene expression and signal transduction, respectively; MTT assays and flow cytometry were used to assess metabolic activity and apoptosis; and mass spectrometry was used to quantify different bioactive sphingolipid intermediates associated with SphK activity. ABC294640 was used in a murine PEL xenograft model to assess the effects of SphK inhibition on KSHV+ lymphoma progression in vivo. Results and Conclusions We find that targeting SphK induces caspase cleavage and apoptosis for KSHV+ patient-derived PEL cells in the presence or absence of co-infection with the Epstein-Barr virus (EBV), whereas uninfected B cell tumor lines are less readily affected. Validating these results, we find that systemic administration of ABC294640 induces tumor regression in the PEL xenograft model. Complimentary ex vivo and in vitro analyses revealed that ABC294640 suppresses constitutive signal transduction associated with proliferation and survival of PEL cells, and increases intracellular accumulation of pro-apoptotic sphingolipid intermediates as well as KSHV lytic genes previously associated with cancer cell death. These results justify additional studies to identify mechanisms for SphK and S1P regulation of virus-associated PEL pathogenesis. Importantly, they also justify evaluation of ABC294640 in clinical trials as a single agent, or in combination with existing approaches, for the treatment of PEL and possibly other malignancies associated with oncogenic viruses. Disclosures: No relevant conflicts of interest to declare.
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An, Ningfei, Yeong-Bin Im, Peng Gao, Luciano J. Costa, Robert K. Stuart, Charles Smith, and Yubin Kang. "Potential Use of Sphingosine Kinase-2 Selective Inhibitors for the Treatment of Multiple Myeloma." Blood 118, no. 21 (November 18, 2011): 5105. http://dx.doi.org/10.1182/blood.v118.21.5105.5105.
Повний текст джерелаАнотація:
Abstract Abstract 5105 Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ∼10,600 deaths annually. Despite the widespread use of several highly active chemotherapy agent (thalidomide, lenalidomide and bortezomib) and the incorporation of autologous hematopoietic stem cell transplantation, MM remains an incurable disease, suggesting the need for a better understanding of the disease's molecular pathways and for the development of novel agents. Sphingolipid metabolism is being increasingly recognized as a key pathway in cancer biology. Among a series of sphingolipid-metabolizing enzymes, sphingosine kinases (SK-1 and -2) are considered to be key regulators of cancer cell proliferation. SK phosphorylates sphingosine to sphingosine-1-phosphate (S1P) and affects the delicate balance between apoptotic ceramide/sphingosine and proliferative S1P. SKs are highly expressed in several solid tumors including pancreatic cancer and ovarian cancer. Currently, however, very little is known about how the SK is expressed in MM, and how sphingolipids respond to drugs targeting SK. We hypothesize that SKs play important roles in the pathogenesis of MM, thus providing a novel target for the treatment of MM. Apogee Biotechnology Corporation has generated new, orally bioavailable small molecule SK inhibitors that have in vitro and in vivo activity in a variety of models of cancer and inflammatory diseases. Several advantages of these SK inhibitors over other anti-MM drugs include: the role of the target in multiple key pathways driving MM; the low toxicity observed in GLP toxicology testing; the oral bioavailability that will simplify administration; and the ability to be combined with the standard drugs for MM (i.e, bortezomib). ABC294640, a SK2-selective inhibitor has recently entered single-agent clinical testing in advanced pancreatic cancer at our institution. In the current study, we investigated the expression patterns of SK2 in myeloma cells and the therapeutic potential of ABC294640 in the treatment of MM. We found that SK2 is highly expressed in several myeloma cell lines and in primary human CD138+ myeloma cells. Compared to the A498 human kidney adenocarcinoma cell line, the expression levels of SK2 mRNA were 1.4–12 fold higher in myeloma cell lines. The expression level of SK2 mRNA was > 62 fold higher in primary human CD138+ myeloma cells, compared to CD138- cells (n = 4). When myeloma cells were treated with ABC294640, cell proliferation was effectively inhibited with IC50 of ∼20 μM, including steroid resistant MM1.R myeloma cells. The degree of cell growth inhibition by ABC294640 correlated well with the expression level of SK2 mRNA in the myeloma cells. We also found that ABC294640 induces PARP cleavage and caspase 3 and 9 activation, indicating that the SK2 inhibitor induces apoptotic cell death. We are currently testing the effects of ABC294640 alone and in combination with dexamethasone or proteasome inhibitors in vivo in the XBP-1s transgenic MM model. Preclinical toxicities of the drug combinations will also be determined in the mouse models. Our studies provide the first evidence of SK2 in the pathogenesis of MM, and suggest excellent therapeutic potential of SK2-selective inhibitors for the treatment of MM. This work is supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award Disclosures: No relevant conflicts of interest to declare.
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Rosell, Rafael, Imane Chaib, Xueting Cai, David Llige, Mariacarmela Santarpia, Eloisa Jantus-Lewintre, Martyna Filipska, et al. "Osimertinib and dihydroartemisinin: A novel drug combination targeting head and neck squamous cell carcinoma." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e17526-e17526. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e17526.
Повний текст джерелаАнотація:
e17526 Background: Recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) has a dismal prognosis with limited progression-free survival and overall survival, even when treated with different combinations of chemotherapy, targeted therapies and immunotherapy. We explored in vitro and in vivo the effect of the epidermal growth factor (EGFR) inhibitor, osimertinib, alone and in combination with dihydroartemisinin (DHA) in HNSCC. Methods: The combination of osimertinib with DHA was tested in the FaDu and CAL27 HNSCC cell lines. Tumor cell proliferation assays were conducted in cultured cells and mouse xenografts. Western blotting analysis of related signal pathways was performed to investigate the molecular mechanisms of the inhibitory effect of DHA and the combination. Other compounds, which inhibit signal transducer and activator of transcription 3 (STAT3), Src-family kinases (SFKs), sphingosine kinase 1 (SPHK1), or the receptor tyrosine kinase (RTK) AXL were also combined with osimertinib in vitro. Results: Osimertinib exerted synergistic cytotoxicity toward FaDu and CAL27 HNSCC cells when combined with DHA. DHA reversed the osimertinib-induced STAT3 and Src, phosphorylation. The double combination inhibited AXL expression. The anticancer potential of osimertinib plus DHA combination was validated in vivo on FaDu and CAL27 xenografts in mice without notable side effects. Conclusions: The results illustrate that the combinatory therapy of osimertinib and DHA, as a repurposing anticancer drug, could be a novel therapeutic strategy for recurrent and/or metastatic HNSCC patients. The findings indicate that a clinical trial is warranted to confirm the benefit of the combination.
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Beyer, Sandra, Stephanie Schwalm, Josef Pfeilschifter, and Andrea Huwiler. "Renal Mesangial Cells Isolated from Sphingosine Kinase 2 Transgenic Mice Show Reduced Proliferation and are More Sensitive to Stress-Induced Apoptosis." Cellular Physiology and Biochemistry 47, no. 6 (2018): 2522–33. http://dx.doi.org/10.1159/000491625.
Повний текст джерелаАнотація:
Background/Aims: Sphingosine 1-phosphate (S1P) is considered as a key molecule regulating various cell functions including cell growth and death. It is produced by two sphingosine kinases (SK) denoted as SK-1 and SK-2. Whereas SK-1 has been extensively studied and has been appointed a role in promoting cell growth, the function of SK-2 is controversial, and both pro-proliferative and pro-apoptotic functions have been suggested. In this study we investigated whether renal mesangial cells isolated from transgenic mice overexpressing the human Sphk2 gene (hSK2-tg) showed an altered cell response towards growth-inducing and apoptotic stimuli. Methods: hSK2-tg mice were generated by using a Quick KnockinR strategy. Renal mesangial cells were isolated by a differential sieving method and further cultivated in vitro. Lipids were quantified by mass spectrometry. Protein expression was determined by Western blot analysis, cell proliferation was determined by 3H-thymidine incorporation, and apoptosis was determined by a DNA fragmentation ELISA. Results: We show here that kidneys and mesangial cells from hSK2-tg mice express the hSK2 as well as the endogenous mouse mSK2. hSK2 and mSK2 predominantly resided in the cytosol of quiescent transgenic cells. However, S1P accumulated strongly in the nucleus and only minimally in the cytosol of transgenic cells. Functionally, hSK2-tg cells proliferated less than control cells under normal growth conditions and were also more sensitive towards stress-induced apoptosis. On the molecular level, this was reflected by reduced ERK and Akt/PKB activation, and upon staurosporine treatment, by a sensitized mitochondrial pathway as manifested by reduced anti-apoptotic Bcl-XL expression and increased cleavage of caspase-9, downstream caspase-3 and PARP-1. Conclusion: Altogether, these data demonstrate that SK-2 exerts an antiproliferative and apoptosis-sensitizing effect in renal mesangial cells which suggests that selective inhibitors of SK-2 may promote proliferation and reduce apoptosis and this may have impact on the outcome of proliferation-associated diseases such as mesangioproliferative glomerulonephritis.
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Grbčić, Petra, Thomas O. Eichmann, Sandra Kraljević Pavelić, and Mirela Sedić. "The Sphingosine Kinase 2 Inhibitor ABC294640 Restores the Sensitivity of BRAFV600E Mutant Colon Cancer Cells to Vemurafenib by Reducing AKT-Mediated Expression of Nucleophosmin and Translationally-Controlled Tumour Protein." International Journal of Molecular Sciences 22, no. 19 (October 5, 2021): 10767. http://dx.doi.org/10.3390/ijms221910767.
Повний текст джерелаАнотація:
Vemurafenib (PLX4032), small-molecule inhibitor of mutated BRAFV600E protein, has emerged as a potent anti-cancer agent against metastatic melanoma harboring BRAFV600E mutation. Unfortunately, the effect of PLX4032 in the treatment of metastatic BRAF mutated colorectal cancer (CRC) is less potent due to high incidence of fast-developing chemoresistance. It has been demonstrated that sphingolipids are important mediators of chemoresistance to various therapies in colon cancer. In this study, we will explore the role of major regulators of sphingolipid metabolism and signaling in the development of resistance to vemurafenib in BRAF mutant colon cancer cells. The obtained data revealed significantly increased expression levels of activated sphingosine kinases (SphK1 and SphK2) in resistant cells concomitant with increased abundance of sphingosine-1-phosphate (S1P) and its precursor sphingosine, which was accompanied by increased expression levels of the enzymes regulating the ceramide salvage pathway, namely ceramide synthases 2 and 6 and acid ceramidase, especially after the exposure to vemurafenib. Pharmacological inhibition of SphK1/SphK2 activities or modulation of ceramide metabolism by exogenous C6-ceramide enhanced the anti-proliferative effect of PLX4032 in resistant RKO cells in a synergistic manner. It is important to note that the inhibition of SphK2 by ABC294640 proved effective at restoring the sensitivity of resistant cells to vemurafenib at the largest number of combinations of sub-toxic drug concentrations with minimal cytotoxicity. Furthermore, the obtained findings revealed that enhanced anti-proliferative, anti-migratory, anti-clonogenic and pro-apoptotic effects of a combination treatment with ABC294640 and PLX4032 relative to either drug alone were accompanied by the inhibition of S1P-regulated AKT activity and concomitant abrogation of AKT-mediated cellular levels of nucleophosmin and translationally-controlled tumour protein. Collectively, our study suggests the possibility of using the combination of ABC294640 and PLX4032 as a novel therapeutic approach to combat vemurafenib resistance in BRAF mutant colon cancer, which warrants additional preclinical validation studies.
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Paugh, Barbara S., Steven W. Paugh, Lauren Bryan, Dmitri Kapitonov, Katarzyna M. Wilczynska, Sunita M. Gopalan, Hanna Rokita, Sheldon Milstien, Sarah Spiegel та Tomasz Kordula. "EGF regulates plasminogen activator inhibitor‐1 (PAI‐1) by a pathway involving c‐Src, PKCδ, and sphingosine kinase 1 in glioblastoma cells". FASEB Journal 22, № 2 (12 вересня 2007): 455–65. http://dx.doi.org/10.1096/fj.07-8276com.
Повний текст джерела
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Prieschl, Eva E., Robert Csonga, Veronica Novotny, Gary E. Kikuchi, and Thomas Baumruker. "The Balance between Sphingosine and Sphingosine-1-Phosphate Is Decisive for Mast Cell Activation after Fc∈ Receptor I Triggering." Journal of Experimental Medicine 190, no. 1 (July 1, 1999): 1–8. http://dx.doi.org/10.1084/jem.190.1.1.
Повний текст джерелаАнотація:
Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and sphingosine-1-phosphate in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids sphingosine and sphingosine-1-phosphate that determines the allergic responsiveness of mast cells. High intracellular concentrations of sphingosine act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen–mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of sphingosine-1-phosphate, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of sphingosine-1-phosphate are dominant over sphingosine as they counteract its inhibitory potential. Therefore, it might be inferred that sphingosine-kinase is pivotal to the activation of signaling cascades initiated at the Fc∈ receptor I by modulating the balance of the counterregulatory lipids.
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Oaks, Joshua, Paolo Neviani, A. Mukhopadhyay, Ramasamy Santhanam, Y. Ma, Charlene Mao, Guido Marcucci, et al. "FTY720 but Not Its Immunosuppressive Phosphorylated Form FTY720-P Exerts Anti-Leukemic Activity towards Ph(+) and Ph(−) Myeloproliferative Disorders through Reactivation of the PP2A Tumor Suppressor." Blood 114, no. 22 (November 20, 2009): 3259. http://dx.doi.org/10.1182/blood.v114.22.3259.3259.
Повний текст джерелаАнотація:
Abstract Abstract 3259 Poster Board III-1 We have shown that the sphingosine analogue FTY720 markedly induces apoptosis of Ph(+) CML (chronic phase and blast crisis) and B-ALL progenitors regardless of their sensitivity to tyrosine kinase inhibitors through the reactivation of the tumor suppressor PP2A (protein phosphatase 2A). Here we report the identification of the molecular mechanism underlying the ability of FTY720 to activate PP2A, and its therapeutic potential towards Ph(+) and Ph(−) myeloproliferative disorders (MPDs). First, we show that the pro-apoptotic and anti-proliferative effect of FTY720 is not limited to BCR/ABL+ leukemias but can be extended to Jak2V617F-driven MPDs that are also characterized by the extensive (80% inhibition) Jak2V617F-mediated suppression of PP2A activity. In fact, shRNA-mediated interference with the hnRNP A1-SET inhibitory pathway in Jak2V617F-expressing erythro-myeloid precursors revealed that Jak2V617F, like BCR/ABL, utilizes these effectors to impair PP2A function in a dose- and kinase-dependent manner. FTY720 treatment (0.5-1mM) fully restored PP2A activity and resulted in decreased BFU-E colony formation of Jak2V617F-transduced Lin- mouse marrow progenitors. Likewise, FTY720 decreased Jak2V617F expression/activity and inhibited clonogenicity of Jak2V617F-expressing 32D-EpoR+, Ba/F3 and TF-1 cells in PP2A/SHP-1-dependent manner. Secondly, we show that PP2A is also inhibited in a SET-dependent manner in CD34+/CD38- stem cells (HSCs) from CML patients. In Ph(+) HSCs, FTY720 but not imatinib or dasatinib decreases the number of long-term culture initiating cells (LTC-IC) and quiescent stem cells (CFSEmax) through activation of BCR/ABL-independent caspase-mediated apoptosis. Importantly, enhanced self-renewal of Ph(+) HSCs seems to depend on constitutive nuclear β-catenin transcriptional activity. In fact, FTY720 or PP2Ac lentiviral transduction, but not imatinib/dasatinib, induces b-catenin inactivation/degradation as measured by in situ immunofluorescence and LET/TCF assays. The effect of FTY720 in Ph(+) HSCs relies on the PP2A ability to override the BCR/ABL-independent inactivation of the β-catenin negative regulator GSK-3β, as treatment with the GSK-3β inhibitors LiCl and SB216763 increased the CFSEmax fraction and hampered the pro-apoptotic effect of FTY720 on quiescent Ph(+) HSCs. Notably, FTY720 did not affect normal stem/progenitor cell viability. Similar results were obtained with the LSK fraction from SCL-tTA/BCR/ABL mice. Reportedly, FTY720 to act as an immunosuppressant requires phosphorylation by sphingosine kinase (SPHK) 2. To determine whether conversion of FTY720 into FTY720-P is important for its anti-leukemic activity in BCR/ABL- and Jak2V617F-expressing cells, we used growth factor-dependent hematopoietic cells transformed by the constitutive activity of these oncogenic tyrosine kinases. In Jak2V617F- and BCR/ABL-transformed cells, PP2A activation by FTY720 (2.5μM, 6h) is not changed when phosphorylation is prevented by treatment with the SPHK inhibitor dimethylsphingosine (2.5μM, 6h). Accordingly, FTY720-P did not activate PP2A and did not affect BCR/ABL- and Jak2V617F-driven colony formation, indicating that the anti-leukemic activity of FTY720 does not require its phosphorylation. Because we have shown that sphingolipid PP2A activator ceramide specifically binds to SET, thus disrupting the SET/PP2A interaction in non hematopoietic cells, we investigated the effect of BCR/ABL or Jak2 V617F and that of FTY720 on ceramide production. LS-MS mass-spectrometry showed that levels of ceramide were similar in untreated and imatinib-treated BCR/ABL+ cells. Similarly, levels of diacylglycerol (DAG) kinase-phosphorylated ceramide were unchanged by treatment with FTY720, suggesting that oncogenic tyrosine kinase-induced suppression of PP2A and FTY720-dependent PP2A reactivation are not ceramide-mediated. Accordingly, FTY720 displaces biotin-labeled C6-ceramide (10μM) from SET, as measured by ceramide affinity chromatography followed by determination of SET levels in the eluted fraction. Thus, FTY720 represents a powerful therapeutic tool as it has the potential to treat and, perhaps, eradicate Ph(+) and Ph(−) MPDs by efficiently inhibiting oncogene-dependent and -;independent signals through the reactivation of the tumor suppressor PP2A via disruption of the SET/PP2A inhibitory complex. Disclosures: Cortes: Novartis: Research Funding. Cancelas:CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.
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Lee, Kiho, Iliana Escobar, Yeeun Jang, Wooseong Kim, Frederick M. Ausubel, and Eleftherios Mylonakis. "In the Model Host Caenorhabditis elegans, Sphingosine-1-Phosphate-Mediated Signaling Increases Immunity toward Human Opportunistic Bacteria." International Journal of Molecular Sciences 21, no. 21 (October 22, 2020): 7813. http://dx.doi.org/10.3390/ijms21217813.
Повний текст джерелаАнотація:
Sphingosine-1-phophate (S1P) is a sphingolipid-derived signaling molecule that controls diverse cellular functions including cell growth, homeostasis, and stress responses. In a variety of metazoans, cytosolic S1P is transported into the extracellular space where it activates S1P receptors in a concentration-dependent manner. In the free-living nematode Caenorhabditis elegans, the spin-2 gene, which encodes a S1P transporter, is activated during Gram-positive or Gram-negative bacterial infection of the intestine. However, the role during infection of spin-2 and three additional genes in the C. elegans genome encoding other putative S1P transporters has not been elucidated. Here, we report an evolutionally conserved function for S1P and a non-canonical role for S1P transporters in the C. elegans immune response to bacterial pathogens. We found that mutations in the sphingosine kinase gene (sphk-1) or in the S1P transporter genes spin-2 or spin-3 decreased nematode survival after infection with Pseudomonas aeruginosa or Enterococcus faecalis. In contrast to spin-2 and spin-3, mutating spin-1 leads to an increase in resistance to P. aeruginosa. Consistent with these results, when wild-type C. elegans were supplemented with extracellular S1P, we found an increase in their lifespan when challenged with P. aeruginosa and E. faecalis. In comparison, spin-2 and spin-3 mutations suppressed the ability of S1P to rescue the worms from pathogen-mediated killing, whereas the spin-1 mutation had no effect on the immune-enhancing activity of S1P. S1P demonstrated no antimicrobial activity toward P. aeruginosa and Escherichia coli and only minimal activity against E. faecalis MMH594 (40 µM). These data suggest that spin-2 and spin-3, on the one hand, and spin-1, on the other hand, transport S1P across cellular membranes in opposite directions. Finally, the immune modulatory effect of S1P was diminished in C. eleganssek-1 and pmk-1 mutants, suggesting that the immunomodulatory effects of S1P are mediated by the p38 MAPK signaling pathway.
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dela Paz, Nathaniel G., Benoît Melchior та John A. Frangos. "Shear stress induces Gαq/11 activation independently of G protein-coupled receptor activation in endothelial cells". American Journal of Physiology-Cell Physiology 312, № 4 (1 квітня 2017): C428—C437. http://dx.doi.org/10.1152/ajpcell.00148.2016.
Повний текст джерелаАнотація:
Mechanochemical signal transduction occurs when mechanical forces, such as fluid shear stress, are converted into biochemical responses within the cell. The molecular mechanisms by which endothelial cells (ECs) sense/transduce shear stress into biological signals, including the nature of the mechanosensor, are still unclear. G proteins and G protein-coupled receptors (GPCRs) have been postulated independently to mediate mechanotransduction. In this study, we used in situ proximity ligation assay (PLA) to investigate the role of a specific GPCR/Gαq/11 pair in EC shear stress-induced mechanotransduction. We demonstrated that sphingosine 1-phosphate (S1P) stimulation causes a rapid dissociation at 0.5 min of Gαq/11 from its receptor S1P3, followed by an increased association within 2 min of GPCR kinase-2 (GRK2) and β-arrestin-1/2 with S1P3 in human coronary artery ECs, which are consistent with GPCR/Gαq/11 activation and receptor desensitization/internalization. The G protein activator AlF4 resulted in increased dissociation of Gαq/11 from S1P3, but no increase in association between S1P3 and either GRK2 or β-arrestin-1/2. The G protein inhibitor guanosine 5′-(β-thio) diphosphate (GDP-β-S) and the S1P3 antagonist VPC23019 both prevented S1P-induced activation. Shear stress also caused the rapid activation within 7 s of S1P3/Gαq/11. There were no increased associations between S1P3 and GRK2 or S1P3 and β-arrestin-1/2 until 5 min. GDP-β-S, but not VPC23019, prevented dissociation of Gαq/11 from S1P3 in response to shear stress. Shear stress did not induce rapid dephosphorylation of β-arrestin-1 or rapid internalization of S1P3, indicating no GPCR activation. These findings suggest that Gαq/11 participates in the sensing/transducing of shear stress independently of GPCR activation in ECs.
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Bandhuvula, Padmavathi, Norman Honbo, Guan-Ying Wang, Zhu-Qiu Jin, Henrik Fyrst, Meng Zhang, Alexander D. Borowsky, Lisa Dillard, Joel S. Karliner, and Julie D. Saba. "S1P lyase: a novel therapeutic target for ischemia-reperfusion injury of the heart." American Journal of Physiology-Heart and Circulatory Physiology 300, no. 5 (May 2011): H1753—H1761. http://dx.doi.org/10.1152/ajpheart.00946.2010.
Повний текст джерелаАнотація:
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes cardiomyocyte survival and contributes to ischemic preconditioning. S1P lyase (SPL) is a stress-activated enzyme responsible for irreversible S1P catabolism. We hypothesized that SPL contributes to oxidative stress by depleting S1P pools available for cardioprotective signaling. Accordingly, we evaluated SPL inhibition as a strategy for reducing cardiac ischemia-reperfusion (I/R) injury. We measured SPL expression and enzyme activity in murine hearts. Basal SPL activity was low in wild-type cardiac tissue but was activated in response to 50 min of ischemia ( n = 5, P < 0.01). Hearts of heterozygous SPL knockout mice exhibited reduced SPL activity, elevated S1P levels, smaller infarct size, and increased functional recovery after I/R compared with littermate controls ( n = 5, P < 0.01). The small molecule tetrahydroxybutylimidazole (THI) is a Federal Drug Administration-approved food additive that inhibits SPL. When given overnight at 25 mg/l in drinking water, THI raised S1P levels and reduced SPL activity ( n = 5, P < 0.01). THI reduced infarct size and enhanced hemodynamic recovery in response to 50 min of ischemia and to 40 min of reperfusion in ex vivo hearts ( n = 7, P < .01). These data correlated with an increase in MAP kinase-interacting serine/threonine kinase 1, eukaryotic translation initiation factor 4E, and ribosomal protein S6 phosphorylation levels after I/R, suggesting that SPL inhibition enhances protein translation. Pretreatment with an S1P1 and S1P3 receptor antagonist partially reversed the effects of THI. These results reveal, for the first time, that SPL is an ischemia-induced enzyme that can be targeted as a novel strategy for preventing cardiac I/R injury.
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Birchwood, Christine J., Julie D. Saba, Robert C. Dickson, and Kyle W. Cunningham. "Calcium Influx and Signaling in Yeast Stimulated by Intracellular Sphingosine 1-Phosphate Accumulation." Journal of Biological Chemistry 276, no. 15 (January 19, 2001): 11712–18. http://dx.doi.org/10.1074/jbc.m010221200.
Повний текст джерелаАнотація:
In mammalian cells, intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways. The budding yeastSaccharomyces cerevisiaeexpresses enzymes that can synthesize and degrade S1P and related molecules, but their possible role in calcium signaling has not yet been tested. Here we examine the effects of S1P accumulation on calcium signaling using a variety of yeast mutants. Treatment of yeast cells with exogenous sphingosine stimulated Ca2+accumulation through two distinct pathways. The first pathway required the Cch1p and Mid1p subunits of a Ca2+influx channel, depended upon the function of sphingosine kinases (Lcb4p and Lcb5p), and was inhibited by the functions of S1P lyase (Dpl1p) and the S1P phosphatase (Lcb3p). The biologically inactive stereoisomer of sphingosine did not activate this Ca2+influx pathway, suggesting that the active S1P isomer specifically stimulates a calcium-signaling mechanism in yeast. The second Ca2+influx pathway stimulated by the addition of sphingosine was not stereospecific, was not dependent on the sphingosine kinases, occurred only at higher doses of added sphingosine, and therefore was likely to be nonspecific. Mutants lacking both S1P lyase and phosphatase (dpl1 lcb3double mutants) exhibited constitutively high Ca2+accumulation and signaling in the absence of added sphingosine, and these effects were dependent on the sphingosine kinases. These results show that endogenous S1P-related molecules can also trigger Ca2+accumulation and signaling. Several stimuli previously shown to evoke calcium signaling in wild-type cells were examined inlcb4 lcb5double mutants. All of the stimuli produced calcium signals independent of sphingosine kinase activity, suggesting that phosphorylated sphingoid bases might serve as messengers of calcium signaling in yeast during an unknown cellular response.
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Miller, Anna V., Sergio E. Alvarez, Sarah Spiegel та Deborah A. Lebman. "Sphingosine Kinases and Sphingosine-1-Phosphate Are Critical for Transforming Growth Factor β-Induced Extracellular Signal-Regulated Kinase 1 and 2 Activation and Promotion of Migration and Invasion of Esophageal Cancer Cells". Molecular and Cellular Biology 28, № 12 (21 квітня 2008): 4142–51. http://dx.doi.org/10.1128/mcb.01465-07.
Повний текст джерелаАнотація:
ABSTRACT Transforming growth factor β (TGFβ) plays a dual role in oncogenesis, acting as both a tumor suppressor and a tumor promoter. These disparate processes of suppression and promotion are mediated primarily by Smad and non-Smad signaling, respectively. A central issue in understanding the role of TGFβ in the progression of epithelial cancers is the elucidation of the mechanisms underlying activation of non-Smad signaling cascades. Because the potent lipid mediator sphingosine-1-phosphate (S1P) has been shown to transactivate the TGFβ receptor and activate Smad3, we examined its role in TGFβ activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and promotion of migration and invasion of esophageal cancer cells. Both S1P and TGFβ activate ERK1/2, but only TGFβ activates Smad3. Both ligands promoted ERK1/2-dependent migration and invasion. Furthermore, TGFβ rapidly increased S1P, which was required for TGFβ-induced ERK1/2 activation, as well as migration and invasion, since downregulation of sphingosine kinases, the enzymes that produce S1P, inhibited these responses. Finally, our data demonstrate that TGFβ activation of ERK1/2, as well as induction of migration and invasion, is mediated at least in part by ligation of the S1P receptor, S1PR2. Thus, these studies provide the first evidence that TGFβ activation of sphingosine kinases and formation of S1P contribute to non-Smad signaling and could be important for progression of esophageal cancer.
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Shu, Xiaodong, Weicheng Wu, Raymond D. Mosteller, and Daniel Broek. "Sphingosine Kinase Mediates Vascular Endothelial Growth Factor-Induced Activation of Ras and Mitogen-Activated Protein Kinases." Molecular and Cellular Biology 22, no. 22 (November 15, 2002): 7758–68. http://dx.doi.org/10.1128/mcb.22.22.7758-7768.2002.
Повний текст джерелаАнотація:
ABSTRACT Vascular endothelial growth factor (VEGF) signaling is critical to the processes of angiogenesis and tumor growth. Here, evidence is presented for VEGF stimulation of sphingosine kinase (SPK) that affects not only endothelial cell signaling but also tumor cells expressing VEGF receptors. VEGF or phorbol 12-myristate 13-acetate treatment of the T24 bladder tumor cell line resulted in a time- and dose-dependent stimulation of SPK activity. In T24 cells, VEGF treatment reduced cellular sphingosine levels while raising that of sphingosine-1-phosphate. VEGF stimulation of T24 cells caused a slow and sustained accumulation of Ras-GTP and phosphorylated extracellular signal-regulated kinase (phospho-ERK) compared with that after EGF treatment. Small interfering RNA (siRNA) that targets SPK1, but not SPK2, blocks VEGF-induced accumulation of Ras-GTP and phospho-ERK in T24 cells. In contrast to EGF stimulation, VEGF stimulation of ERK1/2 phosphorylation was unaffected by dominant-negative Ras-N17. Raf kinase inhibition blocked both VEGF- and EGF-stimulated accumulation of phospho-ERK1/2. Inhibition of SPK by pharmacological inhibitors, a dominant-negative SPK mutant, or siRNA that targets SPK blocked VEGF, but not EGF, induction of phospho-ERK1/2. We conclude that VEGF induces DNA synthesis in a pathway which sequentially involves protein kinase C (PKC), SPK, Ras, Raf, and ERK1/2. These data highlight a novel mechanism by which SPK mediates signaling from PKC to Ras in a manner independent of Ras-guanine nucleotide exchange factor.
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Walker, Christopher, Joshua J. Oaks, Paolo Neviani, Jason G. Harb, Ramasamy Santhanam, Y. Ma, Guido Marcucci, et al. "PP2A Activating Drugs (PAD): Anti-Leukemic and Non-Toxic Activity of Two Novel and Non-Immunosuppressive FTY720 Derivatives." Blood 116, no. 21 (November 19, 2010): 2901. http://dx.doi.org/10.1182/blood.v116.21.2901.2901.
Повний текст джерелаАнотація:
Abstract Abstract 2901 FTY720 is a sphingosine analog proposed by the FDA for treating Multiple Sclerosis patients because of its immunosuppressive activity, which depends on its ability to prevent lymphocyte egress into the peripheral blood. To act as an immunosuppressive drug, FTY720 undergoes sphingosine kinase 2 (SPHK2) phosphorylation and internalization upon interaction with the sphingosine-1-phosphate receptor 1 (S1PR1). FTY720 also acts as a potent activator of protein phosphatase 2A (PP2A), a tumor suppressor found inactivated in chronic and blast crisis CML with wild type or imatinib/dasatinib-resistant BCR-ABL1, Ph+ B-ALL, KitD816V AML, Jak2V617F+ MPDs and other leukemias/lymphomas. FTY720 treatment of cell lines and primary progenitors isolated from bone marrow of patients with these malignancies, markedly suppressed leukemic cell proliferation/survival and induced apoptosis in a PP2A-dependent manner. Notably, long-term treatment with FTY720 of mice carrying these hematopoietic malignancies significantly prolonged survival and restored normal myelopoiesis without exerting any toxic effect in hematopoietic and non-hematopoietic organs. However, in vivo administration of FTY720 strongly, albeit reversibly, decreases the number of circulating B and T lymphocytes. Here we report that a synthetically phosphorylated FTY720 (FTY720-P) is unable to induce neither PP2A activation nor apoptosis of BCR-ABL1-, Jak2V617F-, or KitD816V-expressing myeloid precursors, indicating that FTY720 phosphorylation is dispensable for its anti-leukemic activity. Thus, we functionally characterize two FTY720 derivatives, QC-FTYSM and OSU-2S, which were synthesized as molecules unable to undergo SPHK2 phosphorylation. Treatment (2.5 uM; 24h) of FTY720-sensitive 32D-BCR/ABL cells with QC-FTYSM and OSU2S results in ∼80% and 40%, respectively, more efficient suppression of BCR-ABL1 expression and kinase activity than that observed with FTY720. Moreover, QC-FTYSM, OSU-2S and FTY720 (2.5uM; 0–60h) induce a progressive block of proliferation and marked induction of apoptosis of 32D-BCR/ABL cells. In fact, a 96%, 98%, and 79% decrease in viability is observed after treatment with QC-FTYSM, OSU-2S and FTY720, respectively. Notably, viability of non-transformed myeloid 32Dcl3 cells is not significantly affected by treatment with FTY720 or its derivatives. Consistent with the ability of FTY720 to induce apoptosis through rescue of PP2A activity, phosphatase assays show identical ability of FTY720, QC-FTYSM and OSU-2S to restore PP2A functionality. In fact, comparable and marked decrease in the amount of inactive Y307-phosphorylated PP2Ac was detected in 32D-BCR/ABL cells treated with FTY720 or its derivatives. To formally demonstrate that QC-FTYSM and OSU-2S lack immunosuppressive activity, we first assessed their ability to be internalized upon interaction/association with the S1PR1 receptor. Thus, cells were transduced with a GFP-tagged S1PR1 and treated with either QC-FTYSM, OSU-2S, or, as positive control, FTY720-P. Confocal microscopy revealed that treatment FTY720-P resulted in a strong S1PR1 internalization. Conversely, exposure of the cells to QC-FTYSM and OSU-2S did not alter the S1PR1 membrane localization, indicating that these molecules did not undergo SHPK2 phosphorylation. Further demonstration of the inability of these compounds to act as immunosuppressive molecules was gained upon in vivo administration of a single dose of FTY720, QC-FTYSM or OSU-2C (10 mg/kg) to wild type FVB/N mice. As expected, percentage of B220+/CD19+ circulating B-cells decreased of ∼90% in FTY720-treated animals. Conversely, the percentage of B-cells after exposure to QC-FTYSM and OSU-2S remained unchanged (≤ 1% decrease). Likewise, the number or CD4+ and CD8+ cells also was not affect by treatment with the QC-FTYSM compound. Note that effect of OSU-2S on T-cells and the toxicity profile and anti-leukemic activity of these drugs in healthy animals and mouse models of deadly leukemias (e.g. T315I+ and blast crisis CML and Ph+ ALL) as well as Ph− MPDs are currently being assessed. Altogether our data indicate that QC-FTYSM and OSU-2S represent two potentially powerful and safe drugs which could be introduced in the current therapeutic protocols for different types of hematopoietic and non-hematopoietic malignancies characterized by functional inactivation of the PP2A tumor suppressor. Disclosures: No relevant conflicts of interest to declare.
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Igarashi, Junsuke, Sylvie G. Bernier, and Thomas Michel. "Sphingosine 1-Phosphate and Activation of Endothelial Nitric-oxide Synthase." Journal of Biological Chemistry 276, no. 15 (January 17, 2001): 12420–26. http://dx.doi.org/10.1074/jbc.m008375200.
Повний текст джерелаАнотація:
Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors. Stimulation of EDG receptors by S1P has been shown to activate the endothelial isoform of nitric-oxide synthase (eNOS) in heterologous expression systems (Igarashi, J., and Michel, T. (2000)J. Biol. Chem.275, 32363–32370). However, the signaling pathways that modulate eNOS regulation by S1P/EDG in vascular endothelial cells remain less well understood. We now report that S1P treatment of bovine aortic endothelial cells (BAEC) acutely increases eNOS enzyme activity; the EC50for S1P activation of eNOS is ∼10 nm. The magnitude of eNOS activation by S1P in BAEC is equivalent to that elicited by the agonist bradykinin. S1P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS phosphorylation at Ser1179, a residue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylation site is inactivated shows attenuated S1P-induced eNOS activation. S1P-induced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). By contrast to S1P, activation of G protein-coupled bradykinin B2 receptors neither activates kinase Akt nor promotes Ser1179eNOS phosphorylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S1P and bradykinin may lead to the identification of new points for eNOS regulation in vascular endothelial cells.
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Adebiyi, Morayo G., Yujin Zhang, Kaiqi Sun, Xia Hu, Shusuan Zhao, Hongyu Wu, Jacob Couturier, Dorothy L. Lewis, Edgar T. Walters, and Yang Xia. "Sphingosine-1-Phosphate (S1P) Signaling through S1P Receptor 1 (S1PR1) in Macrophage Contributes to Pain in Sickle Cell Disease." Blood 128, no. 22 (December 2, 2016): 2476. http://dx.doi.org/10.1182/blood.v128.22.2476.2476.
Повний текст джерелаАнотація:
Abstract Sickle cell disease (SCD) is the most common autosomal recessive hemolytic disorder affecting more than 100,000 African-Americans and millions around the world. The severe health complication of patients with SCD is chronic pain, which is the leading cause of SCD patient morbidity and hospitalization. Our proposed research builds on the results from our nonbiased high-throughput metabolomics screen, which revealed erythrocyte sphingosine-1-phosphate (S1P), a bioactive lipid, is significantly elevated in the circulation of humans and mice with SCD. We identified that erythrocyte-sphingosine kinase 1 (Sphk1), the enzyme that generates S1P, promote erythrocyte sickling and the genetic deletion of Sphk1 significantly reduce pain symptoms in SCD mice. S1P is a pleiotropic molecule that signals via five g-protein coupled receptors (GPCRs) known as S1P receptors (S1PR1-5). S1PR1 is widely expressed in many cell types, including neurons and immune cells. We treated SCD mice with FDA approved drug, FTY720, to specifically target S1PR1. We demonstrated that mice had improved mechanical and thermal pain sensitivity. Notably, using flow cytometry, we found that the percentage of macrophages infiltrated to skin and percentage of S1PR1 expression in macrophages in the skin were significantly induced in SCD mice compared to the controls. This implies that elevated S1PR1 in macrophage contributes to chronic pain in SCD mice. To test this possibility, we treated SCD mice with clodronate liposomes to deplete tissue macrophages. We identified that mice had reduced sensitivity to mechanical and thermal stimuli assessments. Altogether, we provide both genetic and pharmacological evidence that elevated plasma S1P signaling via S1PR1 likely on macrophages contributes to chronic pain in SCD. Additionally, S1PR1 is known to mediate pro-inflammatory cytokine secretion in immune cells. We identified that SCD mice have elevated levels of interleukin-6 (IL-6), a pro-inflammatory cytokine, and is likely involved in SCD pain. To study the role of IL-6 in SCD pain, we generated SCD-IL-6-/- mice and observed improved mechanical and thermal pain sensitivity in these mice. From these evidences, we hypothesize that Sphk1 mediate S1P-S1PR1 activation in macrophages and IL-6 induction to promote SCD pain. Our work addresses a knowledge gap regarding an inflammatory mediated mechanism involved in SCD pain and novel therapies to counteract pain in SCD. Disclosures No relevant conflicts of interest to declare.
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Wu, Weicheng, Raymond D. Mosteller, and Daniel Broek. "Sphingosine Kinase Protects Lipopolysaccharide-Activated Macrophages from Apoptosis." Molecular and Cellular Biology 24, no. 17 (September 1, 2004): 7359–69. http://dx.doi.org/10.1128/mcb.24.17.7359-7369.2004.
Повний текст джерелаАнотація:
ABSTRACT Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-κB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria.
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Sukocheva, Olga A., Lijun Wang, Nathaniel Albanese, Stuart M. Pitson, Mathew A. Vadas, and Pu Xia. "Sphingosine Kinase Transmits Estrogen Signaling in Human Breast Cancer Cells." Molecular Endocrinology 17, no. 10 (October 1, 2003): 2002–12. http://dx.doi.org/10.1210/me.2003-0119.
Повний текст джерелаАнотація:
Abstract Current understanding of cytoplasmic signaling pathways that mediate estrogen action in human breast cancer is incomplete. Here we report that treatment with 17β-estradiol (E2) activates a novel signaling pathway via activation of sphingosine kinase (SphK) in MCF-7 breast cancer cells. We found that E2 has dual actions to stimulate SphK activity, i.e. a rapid and transient activation mediated by putative membrane G protein-coupled estrogen receptors (ER) and a delayed but prolonged activation relying on the transcriptional activity of ER. The E2-induced SphK activity consequently activates downstream signal cascades including intracellular Ca2+ mobilization and Erk1/2 activation. Enforced expression of human SphK type 1 gene in MCF-7 cells resulted in increases in SphK activity and cell growth. Moreover, the E2-dependent mitogenesis were highly promoted by SphK overexpression as determined by colony growth in soft agar and solid focus formation. In contrast, expression of SphKG82D, a dominant-negative mutant SphK, profoundly inhibited the E2-mediated Ca2+ mobilization, Erk1/2 activity and neoplastic cell growth. Thus, our data suggest that SphK activation is an important cytoplasmic signaling to transduce estrogen-dependent mitogenic and carcinogenic action in human breast cancer cells.
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Adada, Mohamad M., K. Alexa Orr-Gandy, Ashley J. Snider, Daniel Canals, Yusuf A. Hannun, Lina M. Obeid та Christopher J. Clarke. "Sphingosine Kinase 1 Regulates Tumor Necrosis Factor-mediated RANTES Induction through p38 Mitogen-activated Protein Kinase but Independently of Nuclear Factor κB Activation". Journal of Biological Chemistry 288, № 38 (9 серпня 2013): 27667–79. http://dx.doi.org/10.1074/jbc.m113.489443.
Повний текст джерелаАнотація:
Sphingosine kinase 1 (SK1) produces the pro-survival sphingolipid sphingosine 1-phosphate and has been implicated in inflammation, proliferation, and angiogenesis. Recent studies identified TRAF2 as a sphingosine 1-phosphate target, implicating SK1 in activation of the NF-κB pathway, but the functional consequences of this connection on gene expression are unknown. Here, we find that loss of SK1 potentiates induction of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted; also known as CCL5) in HeLa cells stimulated with TNF-α despite RANTES induction being highly dependent on the NF-κB pathway. Additionally, we find that SK1 is not required for TNF-induced IKK phosphorylation, IκB degradation, nuclear translocation of NF-κB subunits, and transcriptional NF-κB activity. In contrast, loss of SK1 prevented TNF-induced phosphorylation of p38 MAPK, and inhibition of p38 MAPK, like SK1 knockdown, also potentiates RANTES induction. Finally, in addition to RANTES, loss of SK1 also potentiated the induction of multiple chemokines and cytokines in the TNF response. Taken together, these data identify a potential and novel anti-inflammatory function of SK1 in which chemokine levels are suppressed through SK1-mediated activation of p38 MAPK. Furthermore, in this system, activation of NF-κB is dissociated from SK1, suggesting that the interaction between these pathways may be more complex than currently thought.
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Pitson, Stuart M., Paul A. B. Moretti, Julia R. Zebol, Pu Xia, Jennifer R. Gamble, Mathew A. Vadas, Richard J. D'Andrea, and Binks W. Wattenberg. "Expression of a Catalytically Inactive Sphingosine Kinase Mutant Blocks Agonist-induced Sphingosine Kinase Activation." Journal of Biological Chemistry 275, no. 43 (August 15, 2000): 33945–50. http://dx.doi.org/10.1074/jbc.m006176200.
Повний текст джерела
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Hanna, Atef N., Luc G. Berthiaume, Yutaka Kikuchi, David Begg, Sylvain Bourgoin та David N. Brindley. "Tumor Necrosis Factor-α Induces Stress Fiber Formation through Ceramide Production: Role of Sphingosine Kinase". Molecular Biology of the Cell 12, № 11 (листопад 2001): 3618–30. http://dx.doi.org/10.1091/mbc.12.11.3618.
Повний текст джерелаАнотація:
Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-α-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-α, sphingomyelinase, or C2-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C2-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-α effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-α and C2-ceramide. TNF-α, sphingomyelinase, and C2-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-α- and C2-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-α, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for ∼70% of TNF-α-induced stress fiber formation and cytoskeletal reorganization.